Density gradient centrifugation is a well known process for separating particles contained in a liquid sample. In this process, the particles become suspended in a fluid column of progressively increasing density.
Two methods of density gradient centrifugation are in general use. In the method known as the rate zonal technique, a sample solution containing particles to be separated is layered on a pre-formed gradient column. When centrifugal force is applied, the particles sediment through the gradient in separate zones, so that each zone becomes populated by particles of a specific density. In the rate zonal method, it is necessary that the density of the particles be greater than the density of the gradient, and that the centrifugation be terminated before the particles strike the bottom or the wall of the tube.
Another method of density gradient centrifugation is known as the isopycnic technique. In this method, a particle sediments to a point in the centrifuge tube at which the density of the gradient is the same as the density of the particle. Upon reaching this state of equilibrium, the particles will not sediment further along the column irrespective of how much additional centrifugation is carried out.
Currently, tube carrying rotors as well as bowl type zonal rotors are used for making density gradient separations. The following discussion is limited to tube carrying rotors of which there are three main types: swinging bucket rotors, fixed angle rotors and vertical tube rotors. Of these, it is only in the last two types that reorientation of the tube contents takes place; first, when centrifugal force is applied, and again when it is withdrawn. Thus, a gradient consisting of a vertically stacked column of fluid reorients to a horizontally stacked column during centrifugation and then reorients again in a vertical arrangement at the conclusion of centrifugation. As previously stated, reorientation does not occur in swinging bucket type rotors because in such rotors the centrifuge tube is always oriented in the direction of the force field.
A problem which is frequently encountered in making density gradient separations is that remixing of separated particles occurs when the gradient is reoriented. The problem is somewhat more prevalent where vertical tube rotors are used, since their reorientation angle is larger than that of a fixed angle rotor. It has been observed that the cylindrical shape traditionally employed for centrifuge tubes is not ideal to minimize disturbance of a sample during final reorientation. This is so, because shear stresses act on the separation zone when rotation of the gradient takes place in a non-symmetrical envelope. This can be visualized by noting that reorientation in an elongated cylindrical container results in substantial change in zone width and thickness.
Accordingly, the present invention is directed to a sample container used in reorienting gradient centrifugation which overcomes the problems of the prior art.